Chapter 16 Semen Processing, Storage, Thawing, and Handling

    16-1 Importance and properties of semen diluters

    A. Semen diluter = Semen extender; Diluted semen = Extended semen

    B. Importance of semen dilution:

      a. To increase volume of an ejaculate of semen
      b. To protect sperm during cooling and extend sperm life

    C. Properties of good semen diluters

      a. To be isotonic with semen: 2.9% sodium citrate dihydrate
      b. To have buffering capacity : isotonic sodium citrate solution
      c. To protect sperm from cold shock injury during cooling from body temperature to 5¡É: lecithin and lipoproteins from egg yolk or milk
      d. To provide nutrients for sperm metabolism: egg yolk, milk and some simple sugars
      e. To control microbial contaminants : antibiotics
      f. To protect sperm from injury during freezing and thawing: glyserol
      g. To preserve sperm life with a minimum drop in fertility: combination of known and unknown factors

    16-2 Buffer solutions used in semen diluters

    A. To prevent changes in pH of semen by neutralizing lactic acid produced by metabolic activity of sperm

    B. To provide isotonic environment for sperm

    C. To give no toxicity to sperm at the level required for isotonicity

    16-2-1 Phosphate buffer solution

    A. Preparation:

    Na2HPO4.12H2O: 2.0 g
    KH    2PO4 : 0.2 g

    Distilled H2O: add to make 100 ml solution

    B. Uses: Not been popular because of opaque mixture when added to egg yolk, resulting in poor sperm visibility

    16-2-2 Citrate buffer solution

    A. Preparation:

    Na3C6H5O7.2H2O: 2.9 g

    Distilled H2O : add to meke to 100 ml solution

    B. Uses: Extensively used because of good visibility of sperm when mixed with egg yolk Glycerol should be added after semen was precooled to 5¡É

    16-2-3 Tris Buffer solution

    A. Prepartion:

      Tris(hydroxymethyl)aminomethane : 3.028 g = to be 0.2 mol.
      10% citric acid : add to have pH 6.5
      Fructose : 1.0 g
      Distilled H2O : add to make 100 ml solution

    B. Uses: Extensively used in bull and boar semen dilution. Glycerol may be added before semen is precooled

    16-3 Antimicrobial agents for semen diluters

    A. Microbial organisms in semen:

      a. Pathogenic organisms: See TABLE 16-1; Dramatic efect due largely to Vibrio fetus infection in low fertility bulls
      b. Non-pathogenic organisms: Adverse effects due to their competition with sperm for nutrients; Microbial contamination will be reduced by proper cleaning sheath and under line of male prior to collection and using sterile equipments

    B. Antibiotics used:

      Penicillin : 1000 IU/ ml
      Streptomycin: 1000 ug/ ml
      Polymixin B : 1000 ug/ ml

    16-4 Effective diluters for bull semen

    16-4-1 Yolk-phosphate

    A. Phosphate buffer: egg yolk = 1: 1

    B. Phosphate ion + fat globules of yolk ¡æ opaque mixture ¡æ sperm not visible under microscope

    16-4-2 Yolk-Citrate

    A. For liquid semen: citrate buffer: egg yolk = 1: 1

    B. For frozen semen: citrate buffer: egg yolk = 4: 1

    C. Standard diluter

    16-4-3 Tris buffered-Yolk

    A. Tris buffer: egg yolk = 4: 1

    B. Glycerol can be incorporated into initial diluter without cooling to 5¡É

    C. Equilibration time does not semm to be critical¡¡

    16-4-4 Whole homogenized milk and skim milk

    A. Milk heated to normal pasteurization temperature contains spermicidal material LACTENIN

    B. Heating milk to 90 - 95¡É for 10 min. inactivates lactenin

    C. Whole milk has poor sperm visivility under microscope by light refraction by the fat globules in homogenized milk

    16-4-5 Other diluters: See TABLE 16-2

    A. Addition of simple sugars, amino acids and/or enzymes

    B. Various concentrations of egg yolk

    C. CO2 saturation etc.

    16-5 Processing (frozen) bull semen

    A. Processing steps of frozen bull semen: Dilution¡æ cooling¡æ glycerolation & equilibration¡æ packaging¡æ freezing

    B. Control of semen temperature: With placing semen in warm water bath

    16-5-1 Semen dilution

    A. Predilution: Warm semen with 3 to 4 volumes of diluter tempered in the same water bath ¡æ Lecithin and lipoprotein from yolk of diluter: Protect sperm from cold shock and prevent changes in cell wall permeability during cooling process

    B. Dilution: After cooled to 5¡É, the prediluted semen is diluted to final volume with diluter cooled to 5¡É

    16-5-2 Dilution rate

    A. Optimum number of motile sperm per breeding unit at the time of insemination: 10 million sperm (7-8 million sperm for high fertility bulls and 15 million for low fertility bulls), and % sperm with post-thaw intact acrosome may be involved in determining optimum sperm number per breeding unit

    B. Post-thaw sperm motility of an individual bull: can be determined by post-thaw evaluations on several ejaculates of the bull

    C. Breeding units = semen volume x sperm concentration x post-thaw sperm motility / 10 million

    D. Dilution rate = Total volume of diluted semen (total volume of diluter added + semen volume) = Breeding units x diluted semen volume/ breeding unit (0.5 ml for 0.5 ml straw packaging)

    16-5-3 Cooling semen

    Optimum cooling rate: From body temperature to 5¡É for 1.25 to 2 hours (fast cooling) or for 2 to 4 hours (slow cooling)

    16-5-4 Glycerolation and equlilibration

    A. Causes of freezing damage: From selective freezing of free H2O both inside and outside the sperm cell

      a. Concentrating other cell constituents and solutes outside the cell
      b. Upsetting vital cell components
      c. Changes in cell membrane permeability
      d. Preferential leakage of solutes
      e. pH changes in nonelectrolytes

    B. Sperm protection from freezing by glycerolation: Because glycerol binds water, glycerolation results in:

      a. Decreased freezing point of solutions
      b. Less ice formed
      c. Correspondingly decreased concetration of solutes
      d. Partially dehydrated sperm cells
      e. Reduced selective freezing of water

    C. Other cryopreservatives:

      a. Dimethyl sulfoxide(DMSO): Has a salt-buffering capacity and enters the sperm cells rapidly: Most effective with slow freezing
      b. Glycols and other saccharides

    D. Optimum final level of glycerol added:

      a. Yolk-citrate and tris buffered-yolk diluters: 7%
      b. Milk diluters: 10 - 13%

    E. Temperature at glycerolation: 5¡É in yolk-citrate and milk diluters; Glycerol damage when added at 35¡É; No temperature problem in glycerolation for tris buffered-yolk diluter

    F. Procedure for glycerolation:

      a. Divide diluter into Part I and Part II of equal volume
      b. Part I: no glycerol and semen will be prediluted with Part I
      c. Part II: glycerol added at twice the level desired in the final mixture = 14% glycerol for yolk-citrate or tris buffered-yolk diluter and 20-25% for milk diluter
      d. Prediluted semen will be diluted with Part I remainder and then further diluted by dripping slowly with Part II: 10%, 20%, 30% and 40% in the interval of 20 min.

    G. Equilibration:

      a. Equilibration time: needed for the sperm and diluter to completely mix: at least 1 hour
      b.Temperature during equilibration: 5¡É
      c. Additional research is needed for optimum equilibration time

    16-5-5 Semen packaging: See Figure 16-3

    A. Glass ampoules were used until about 1970

    B. Plastic straws:

      a. Size: 0.5 ml with 113 mm long and 2.8 mm in diameter (In Europe 0.25 or 0.3 ml straws are used)

      b. Straw preparation: One end of straw contains 3-parts plug: Cotton- Polyvinyl alcohol powder- Cotton; This PVA powder allow air to pass through until aqueous material (the semen) comes in contact with the powder and vacuum is used to siphon the diluted semen into the straw and when the fluid reaches the powder, it forms a gel and seals. Heat may be used to fuse the plastic open end.

      c. Storage of straws in liquid nitrogen container: Placed in goblets within a cane after labelled to identify the donor bull and the semen producing business

    16-5-6 Freezing

    A. Freezing procedure: A single layer of straws are placed on a tray at 5.5 cm above the liquid nitrogen level; Straws will reach the temperature of liquid nitrogen vapor in about 2 min.

    B. Optimum freezing rate: See TABLE 16-3:

    -126¡É/min to -7¡É/ min.: satisfactory results: influenced by the type of package, glycerol level, thawing rate and diluter composition

    16-6 Storage and handling of bull semen

    A. Optimum temperature for semen storage:

      a. Liquid semen: at 5¡É
      b. Frozen semen: below -75¡É

    NR rate from frozen semen preserved at -79¡É(with dry ice) for 6 months: 13% decreased compared with frozen semen for 1 month

    B. Semen storage in liquid nitrogen container:

      a. Double wall stainless steel or aluminum container with a vacuum between the walls: See FIGURE 16-4: Large storage unit and small field unit
      b. Field unit:
      Liquid nitrogen capacity: 20 liters
      Holding time: 90 days
      Storage capcity: 1,200 straws of 0.5 ml capacity
      Recharging interval: 60 days
      Canister, cane and goblets: goblets should always be filled with liquid nitrogen

    C. Precautions in handling semen

      a. Temperature above -79¡É can be detrimental to sperm survival and the temperature of small straw can be changed very rapidly when exposed to ambient temperatures of 25 to 30¡É
      b. Top of canister should remain 3 to 5 cm below the top of nitrogen tank when looking for semen from a particular bull
      c. Use a particular cane and goblet for a particular bull semen
      d. Use specially designed forceps for lifting straws from goblets

    16-7 Processing boar, ram, stallion and buck semen

    16-7-1 Processing boar semen

    A. Filter ejaculate through 2 layers of cheese cloth to remove gel portion

    B. Place semen in water bath at 38¡É

    C. Evaluate the sample

    D. Liquid semen:

      a. Dilute semen to have a final concentration of 4 to 5 x 109 motile sperm/ 50 ml
      b. Cool diluted semen to 5¡É in 2 hrs.
      c. Normal fertility remained for approx. 3 days
      d. Extensively used in Denmark (semen transportation: within 6 hrs.)

    E. Frozen semen:

      a. Pellet freezing: See Ref. Figure 11.5. of Pursel (1979). Page 151 in H.W. Hawk ed. Animal Reproduction and TABLE 16-4

      b. Straw packaging: Almlid and Johnson(1988) J. Anim. Sci. 66: 2899: With maxi-straws (6 mm o.d.) and 3 % final glycerol concentration: Results of post-thaw motility: 34- 50%, normal apical ridge: 48-57% and plasma membrane integrity: 45%

    16-7-2 Processing ram semen

    A. Diluter: See TABLE 16-5

    B. Dilution rate: 1: 3

    C. Total number of sperm and semen volume/ insemination: 100 million to 500 million in 0.1- 0.3 ml

    D. Semen deposition: In posterior end of cervix

    E. Preservation of semen:

      a. Liquid semen: within 24 hours at 5¡É
      b. Frozen semen: not extensively used with conception rates of 20 to 70%

    F. Water from fluids of female tract enter sperm faster than glycerol can exit, resulting in swelling of sperm head

    16-7-3 Processing stallion semen

    A. Diluter: See TABLE 16-6; cream-gelatin diluter gave higher CR %

    B. Recommended number of motile sperm and semen volume/ insemination: 500 million in 10 to 30 ml

    C. Preservation:

      a. Liquid semen: for 24 to 48 hrs at 5¡É
      b. Froozen semen: Not practical ¡¡

    16-7-4 Processing buck semen

    A. An enzyme in buck seminal plasma interact with egg yolk which produce a toxin capable of killing sperm

    B. Washing buck sperm with Ringer solution is recommended before dilution

    C. Recommended number of motile sperm and semen volume/ insemination: 100 million in 0.25 ml

    D. Diluter: similar to diluter for bull semen

    E. Frozen semen: Satisfactory post-thaw motility by freezing method for bull semen in our laboratory with 3.5% final glycerol concentration

    F. CR % with frozen semen: 40 to 65 %